Kit and Method for Detecting Porous Dental Hydroxyapatite

ABSTRACT

The present invention relates to a kit and a probe for detecting porous dental hydroxyapatite, comprising a protein capable of binding porous dental hydroxyapatite or a detector thereof. The invention also relates to a method for detecting a condition involving porous dental hydroxyapatite comprising detecting in or on a tooth or a sample of the tooth of a subject a protein bound to porous dental hydroxyapatite. The invention also relates to methods for detecting a hypomineralisation developmental dental defect or detecting intact and/or broken MIH enamel, and to a kit and method for removing a protein bound to porous dental hydroxyapatite.

FIELD

The invention relates to a kit and a probe for detecting porous dentalhydroxyapatite and a method for detecting a condition involving porousdental hydroxyapatite.

BACKGROUND

The resilience of teeth depends on a complex interplay between mineral(termed hydroxyapatite) and organic components (proteins, cells andtissues). Under normal conditions the hydroxyapatite in enamel anddentine is organised into an extraordinarily dense structure thatconfers the hardness and toughness required for maintenance of thetooth's integrity. Loss of mineral-density in enamel and dentine resultsin abnormally porous hydroxyapatite, which compromises the tooth'sphysical resilience and can lead to structural failure. Poroushydroxyapatite is caused by several prevalent conditions, includingdental caries and developmental dental defects (DDD).

Dental caries (tooth decay) is a disease caused by bacteria that secreteacid. The acid produced by cariogenic bacteria can dissolvehydroxyapatite in a process termed demineralisation. The initial processof demineralisation (termed incipient caries) leads to discrete regionsof porous hydroxyapatite termed white spot lesions. Over time, awhite-spot lesion may progress to a cavity (i.e. loss of tooth material)or it may stall (termed inactive caries) and re-form a densehydroxyapatite shell in a process called remineralisation. Before acavity forms, the process is reversible (i.e. remineralisation), butonce enamel is lost it cannot be regenerated.

Caries is diagnosed by a combination of visual inspection, physicalchallenge (e.g. scratching with dental probe), and X-ray radiography (todetect caries between teeth or beneath the gum line). Worryingly, thesediagnostic approaches miss approximately half of early caries, and up to13% of teeth diagnosed as carious with these methods are in factcaries-free. Recent attempts at improving diagnosis include use ofequipment that measures electrical impedance, quantitative light-inducedfluorescence (QLF) and infrared laser fluorescence (DIAGNOdent®), butnone have found widespread use because of the cost and size ofapparatus, and problems with inter-individual variation. Anotherapproach has been the use of dyes to detect dental caries in dentine.However, these dyes are not selective for porous hydroxyapatite: theybind to proteins (presumed to be associated with infecting bacteria indentine) or they occupy interstitial space, which reduces specificityand sensitivity. Moreover, these dyes cause the oral cavity to becomediscoloured, bind to healthy teeth, or require visualisation with anirradiator.

There are two main treatments for caries, the selection of which isdictated by the extent of disease. White spot lesions may be treatedwith remineralisation approaches (e.g. fluoride therapy or amorphouscalcium phosphate stabilised with bioactive molecules). Cavities requireconventional restorative dentistry (i.e. fillings).

DDD are another common cause of porous hydroxyapatite. They aredisturbingly prevalent and costly, potentially afflicting over 50% ofthe population with multiple burdens including dental pain,disfigurement and increased caries risk. The two most prevalent DDD aredental fluorosis (characterised by diffuse opacities) and Molar/IncisorHypomineralisation (MIH; characterised by demarcated opacities); bothare caused by environmental agents (i.e. acquired defects). Anotherserious but rare DDD that can result in porous hydroxyapatite is thegenetic disease amelogenesis imperfecta.

MIH typically affects 10-20% of children and is a major risk factor forcaries, a risk factor for orthodontics, and is costly to society. MIH isthought to result from a multifactorial systemic disturbance of theenamel-forming cells. However, other than being dissociated fromfluoride and linked to illness during infancy, the cause of MIH remainsa mystery.

There are currently no products available that are designed to diagnoseand repair MIH or other DDD. Differential diagnosis of caries andvarious DDD can be difficult and is largely dependent upon theexperience and skill of individual dental health professionals. Currentprocedures and/or products developed for remineralisation of caries donot work well on MIH. Restorative treatment is frequently compromisedbecause MIH enamel is soft, porous and poorly delineated from normaltooth tissue.

Accordingly, a need exists for new tools to diagnose, delineate andrepair porous dental hydroxyapatite caused by caries and DDD. Here weaddress this need by detailing new technologies based on our recentdiscoveries of pathogenic mechanisms in conditions involving poroushydroxyapatite.

SUMMARY

A first aspect provides a kit, when used for detecting porous dentalhydroxyapatite, comprising: a protein capable of binding porous dentalhydroxyapatite; or a detector that detects said protein bound to porousdental hydroxyapatite.

A second aspect provides a probe, when used for detecting porous dentalhydroxyapatite, comprising: a protein capable of binding to porousdental hydroxyapatite; and a reporter.

A third aspect provides a method for producing the probe of the secondaspect comprising linking (i) a protein capable of binding to porousdental hydroxyapatite and (ii) a reporter.

A fourth aspect provides a method for detecting a condition involvingporous dental hydroxyapatite comprising detecting in or on a tooth or asample of the tooth of a subject a protein bound to porous dentalhydroxyapatite.

A fifth aspect provides a method for detecting a hypomineralisation DDDcomprising detecting a protein whose concentration bound to testhydroxyapatite of a tooth or of a sample of the tooth is increasedrelative to its concentration bound to control hydroxyapatite of acontrol tooth or of a control sample of a tooth, and detectingamelogenin whose concentration bound to the test hydroxyapatite is nearthat bound to the control hydroxyapatite.

A sixth aspect provides a method for detecting intact and/or broken MIHenamel comprising detecting albumin and hemoglobin bound to MIHhydroxyapatite, wherein detection of albumin but not hemoglobin isindicative of intact MIH enamel, and wherein detection of hemoglobin isindicative of broken MIH enamel.

A seventh aspect provides a kit for removing a protein bound to porousdental hydroxyapatite comprising: (a)(i) one or more washing solutionsor (ii) dry components to prepare one or more washing solutions uponadmixture with water, wherein the one or more washing solutions areadapted to remove a protein bound to porous dental hydroxyapatite; and(b) a remineralisation agent or remedial mineralisation agent.

An eighth aspect provides a method for removing a protein bound toporous dental hydroxyapatite comprising washing a tooth or a sample ofthe tooth with one or more washing solutions.

A ninth aspect provides a kit for removing a protein bound to porousdental hydroxyapatite comprising: one or more washing solutions; or oneor more dry components to prepare one or more washing solutions uponadmixture with water, wherein the one or more washing solutions areadapted to remove a protein in or on a tooth or a sample of the toothdetected as having porous dental hydroxyapatite by the method of thefourth aspect.

The kit, probe or methods of the first to sixth aspects allow detectionin situ or diagnosis ex situ.

The kit, probe or methods of the first to sixth aspects are useful indetecting dental caries and/or MIH/DDD and delineating carious and/orMIH/DDD boundaries in preparation for restoration of a tooth. Theclinician may then specifically remove the carious or MIH tissue thusrevealed, ensuring clean border preparation and improving the likelihoodof restoration success.

The kit of the first aspect or the probe of the second aspect provideskey tools and the method of the fourth aspect allows for routinescreening for porous dental hydroxyapatite. Moreover, the kit, probe ormethods of the first to sixth aspects may be used for early detection ofexposed dental hydroxyapatite. In this manner, the kit, probe or methodsof the first to sixth aspects may be used for routine screening ofdental changes that, without detection, may ultimately lead to dentalcaries (a precursor to caries), enabling accurate and timely targetingof restoration and/or remineralisation to prevent caries progressionand/or promote remineralisation. In some embodiments, the kit, probe ormethods of the first to sixth aspects are particularly suited to routinescreening of children after eruption of the first permanent molar. Itfollows that the kit, probe or methods of the first to sixth aspects arealso suited to routine screening of teeth for early detection of poroushydroxyapatite. Routine regular screening provides an excellentopportunity to detect at the earliest practical moment dental changesthat may lead to dental caries.

Furthermore, the kit, probes and methods of the first to sixth aspectsalso allow monitoring of any treatment, such as known remineralisationtherapies including fluoride or amorphous calcium phosphate that may bestabilised with bioactive molecules, which may be undertaken.

The method of the eighth aspect and the kits of the seventh and ninthaspects enable gentle and/or specific removal of excess proteins thatare strongly retained on porous hydroxyapatite, for example in MIHlesions, to be used prior to or during remineralisation treatments.

The protein of any one of the first to fifth or seventh to ninth aspectsmay be selected from the group: Serum albumin; Complement C3 beta chain;Alpha-1-antitrypsin; Protein S100-A9; Lactotransferrin; Leukocyteelastase inhibitor; Antithrombin-III; Hemoglobin subunit alpha;Hemoglobin subunit beta; Hemoglobin subunit delta; Prolactin-inducibleprotein; Alpha amylase 1; Ig kappa chain V-III region SIE; Ig alpha-2chain C region; Uncharacterized protein c6orf58; and Serpin B3.Furthermore, the protein of any one of the first to fourth or seventh toninth aspects may be Amelogenin.

The kits of the first, seventh and ninth aspects, or the probe of thesecond aspect, may be in alternative forms. One form designates eithersuitability for or restriction to a specific use and is indicated by theword “for”. Another form is restricted to a specific use only and isindicated by the words “when used for”.

The methods of the third to sixth or eighth aspects may be presented inalternative forms, for example in European form (“agent for use”) orsecond medical use (Swiss) form (“use of an agent in the manufacture ofa medicament”).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 plots the protein content of MIH enamel, which is abnormally highrelative to normal enamel. Acid-insoluble proteins were extracted fromnormal enamel (normal) and a group of severe lesions exhibitingpost-eruptive breakdown (specimens 7-11) then quantified bydensitometric dot-blot analysis. Mean values (±SD) are shown forduplicate assays, each done at varied loads to ensure quantitativelinearity (r²>0.95). As indicated, all MIH specimens differedsignificantly from normal when compared pairwise using Student's t-test(homoscedastic, two-tailed). An albumin standard was used to deriveabsolute protein levels from these data.

FIG. 2 illustrates that intact and broken MIH lesions have distinctprotein profiles. Acid-insoluble proteins from MIH lesions and normalenamel (normal) were subjected to SDS-PAGE and stained with CoomassieBlue or immunoblotted with amelogenin antibodies (anti-AMG) asindicated. (A) Comparison of intact-surface and broken-surface lesions(specimens 1-6 and 7-11, respectively), showing distinct patterns forthe major protein bands. The positions of albumin and haemoglobin areindicated (Alb, Hb). (B) Comparison of MIH specimens withsecretion-phase enamel matrix from rat, which served as a control forpredominantly intact amelogenins (AMG). Specimens 7 and 11 arerepresentative of lesions with low or appreciable amounts of amelogeninfragments respectively. For quantification, cross-immunoreactivitybetween rat and human amelogenins was normalized using a humanamelogenin standard (from Abnova, Taipei City, Taiwan). (C) Profiles fortwo intact lesions, comparing the first gel run using fresh extractswith a second run after storage of the same SDS-extracts for 16 wk at−20° C. Note disappearance of the major bands at 66 kDa (albumin).

FIG. 3 lists the results of proteomic analysis of intact and broken MIHlesions, which reveals numerous body fluid proteins in MIH enamel. Theindicated major gel bands from intact and broken lesions (FIG. 2A,specimens 1-11) were subjected to proteomic identification, asdocumented more fully in Table 1. The figure depicts the proteinsidentified in each band, and the specimens in which theseidentifications were made (specimen numbers in parentheses). Gel lanesfor specimens 6 and 7 are reproduced from FIG. 2A to illustrate intactand broken lesions, respectively.

FIG. 4 depicts mineralisation assays revealing that surface integrityregulates the protein composition of MIH enamel. (A) Comparativeprofiling of MIH enamel and body fluids, showing similarities for intactlesions vs. serum and for broken lesions vs. saliva and erythrocytes.(B) Hydroxyapatite-binding (HAp-affinity) assay, showing that a subsetof proteins from mock oral fluid (O-Fluid) were preferentially retained(cf. differences between the Load, Bound and Unbound fractions). Note astrong resemblance between the Bound profile and the broken lesion inpanel A (specimen 7). (C) An equivalent mineral-binding assay to B, butwith powdered MIH enamel in place of hydroxyapatite. The profiles showenamel from an intact lesion, before and after exposure to mock oralfluid (+/−O-Fluid). Note a resemblance of the protein-bound profile (+)to those of broken lesions and hydroxyapatite in panels A and B. Thisresult indicates that loss of gross structure (including intact surface)leads to a marked change in the protein-binding capability of intactlesions. To legitimise these comparisons, both affinity matrices(particulate hydroxyapatite, MIH enamel) were mortar-ground to equalconsistencies (coarse powder) before assay.

FIG. 5 depicts a hydroxyapatite-binding assay (Coomassie-stainedSDS-PAGE) showing that hemoglobin and albumin from mock oral fluid arebound by hydroxyapatite. A three-step washing procedure comprisingwashing sequentially in each of 5 mM MgCl₂, 1 M MgCl₂, and 0.4 M NaH₂PO₄each for 5 min removed >90% of protein from hydroxyapatite.

FIG. 6 provides an amino acid sequence for Human Serum albumin (SEQ IDNO: 1; SwissProt accession P02768).

FIG. 7 provides an amino acid sequence for Human Complement C3 (SEQ IDNO: 2; SwissProt accession P01024).

FIG. 8 provides an amino acid sequence for Human Alpha-1-antitrypsin(SEQ ID NO: 3; SwissProt accession P01009).

FIG. 9 provides an amino acid sequence for Human Protein S100-A9 (SEQ IDNO: 4; SwissProt accession P06702).

FIG. 10 provides an amino acid sequence for Human Lactotransferrin (SEQID NO: 5; SwissProt accession P02788)

FIG. 11 provides an amino acid sequence for Human Leukocyte elastaseinhibitor (SEQ ID NO: 6; SwissProt accession P30740).

FIG. 12 provides an amino acid sequence for Human Antithrombin-III (SEQID NO: 7; SwissProt accession P01008).

FIG. 13 provides an amino acid sequence for Human Hemoglobin subunitalpha (SEQ ID NO: 8; SwissProt accession P69905).

FIG. 14 provides an amino acid sequence for Human Hemoglobin subunitbeta (SEQ ID NO: 9; SwissProt accession P68871).

FIG. 15 provides an amino acid sequence for Human Hemoglobin subunitdelta (SEQ ID NO: 10; SwissProt accession P02042).

FIG. 16 provides an amino acid sequence for (Human Prolactin-inducibleprotein SEQ ID NO: 11; SwissProt accession P12273).

FIG. 17 provides an amino acid sequence for Human Alpha-amylase 1 (SEQID NO: 12; SwissProt accession P04745).

FIG. 18 provides an amino acid sequence for Human Ig kappa chain V-IIIregion SIE (SEQ ID NO: 13; SwissProt accession P01620).

FIG. 19 provides an amino acid sequence for Human Ig alpha-2 chain Cregion (SEQ ID NO: 14; SwissProt accession P01877).

FIG. 20 provides an amino acid sequence for Human Uncharacterizedprotein C6orf58 (SEQ ID NO: 15; SwissProt accession Q6P5S2).

FIG. 21 provides an amino acid sequence for Human Serpin B3 (SEQ ID NO:16; SwissProt accession P29508).

FIG. 22 provides an amino acid sequence for Human Amelogenin, X isoform(SEQ ID NO: 17; SwissProt accession Q99217).

FIG. 23 provides an amino acid sequence for Human Amelogenin, Y isoform(SEQ ID NO: 18; SwissProt accession Q99218).

FIG. 24 provides an amino acid sequence for Mouse Amelogenin (SEQ ID NO:19; SwissProt accession P63277) also corresponding to recombinant MouseAmelogenin.

FIG. 25 provides an amino acid sequence for Bovine Hemoglobin subunitalpha (SEQ ID NO: 20; SwissProt accession P01966).

FIG. 26 provides an amino acid sequence for Bovine Hemoglobin subunitbeta (SEQ ID NO: 21; SwissProt accession P02070).

FIG. 27 depicts the chemical reaction for production of amaleimide-activated coloured reporter through reaction ofN-hydroxysuccinimide ester (SMCC) with amido black (primary amine).Maleimide-activated coloured reporter is sulfhydryl-reactive, ready forconjugation with cysteine-thiols of hemoglobin β subunits.

FIG. 28 depicts the chemical reaction for production of a probe, in thisexample a coloured reporter-conjugated protein, via reaction of amaleimide-activated coloured reporter according to FIG. 28 with cysteinethiol groups (SH) of hemoglobin β subunits. Each hemoglobin tetramerbinds two coloured reporter molecules, and leaves two subunitsunmodified, which is likely important for preserving hemoglobin'shydroxyapatite-binding function.

FIG. 29 depicts the in vitro binding to hydroxyapatite of a probeproduced according to FIGS. 27 and 28 and Example 2. The probe comprisedhemoglobin (Hb), a black-blue coloured reporter (amido black) and alinker. Within 5 min of applying the probe, hydroxyapatite changed todark blue. The probe withstood washing in water, whereas colouredreporter only (i.e. not linked to Hb) was removed by washing in water.The probe was removed from hydroxyapatite by a three-step washingprocedure comprising washing sequentially in each of 5 mM MgCl₂, 1 MMgCl₂, and 0.4 M NaH₂PO₄ each for 5 min.

FIG. 30 depicts the results of Example 3 that demonstrate specificbinding of a probe produced according to Example 2 to porous dentalenamel.

FIG. 31 depicts the results of Example 4 that demonstrate that a probeproduced according to Example 2 can specifically detect earlydemineralisation of surface enamel (model of incipient caries).

FIG. 32 depicts the results of Example 5 that demonstrate that themechanism of action of a probe produced according to Example 2 ishydroxyapatite affinity.

FIG. 33 depicts the results of Example 6 that demonstrate that a probeproduced according to Example 2 specifically labels hypomineralisedenamel and abnormal dentine. Normal enamel and dentine were unlabelled.Hypomineralised enamel was specifically and uniformly labelled anintense violet colour. Abnormal dentine was specifically and uniformlylabelled a deep green colour.

FIG. 34 depicts the results of Example 7 that demonstrate that a probeproduced according to Example 2 can be used to guide removal ofhypomineralised enamel.

FIG. 35 depicts the results of Example 8 that demonstrate that a probeproduced according to Example 2 can be used to guide removal of abnormaldentine.

FIG. 36 depicts the results of Example 9 that demonstrate that detectionof abnormal dentine according to Example 8 can be improved using ableach wash.

FIG. 37 depicts the results of Example 10 that demonstrate that theprobe can be radio-opaque, which can be achieved by substituting theblue chromophore (amido black) of Example 2 foramino-2,4,6-triiodoisophthalic acid (³I).

FIG. 38 depicts the results of Example 11 that demonstrate the relativeeffectiveness of washing solutions comprising Mg²⁺ or PO₄ in removingproteins bound to pure hydroxyapatite.

FIG. 39 depicts the results of Example 12 that demonstrate the relativeeffectiveness of separate or sequential application of washing solutionscomprising Mg²⁺ or PO₄ in removing proteins bound to purehydroxyapatite.

FIG. 40 depicts the results of Example 13 that demonstrate the relativeeffectiveness of combined application of a washing solution comprisingMg²⁺ and PO₄ in removing proteins bound to pure hydroxyapatite.

FIG. 41 depicts the results of Example 14 that demonstrate thatapplication of washing solutions comprising Mg²⁺ or PO₄ removes proteinsfrom hypomineralised enamel, although with reduced efficacy comparedwith the hydroxyapatite model of Examples 11 to 13.

FIG. 42 depicts the results of Example 15 that demonstrate that theefficacy of washing solutions comprising Mg²⁺ or PO₄ in removingproteins from hypomineralised enamel can be improved compared withExample 14 by extending the application period such that the proteinscan be removed quantitatively.

DETAILED DESCRIPTION

Disclosed herein are kits, probes and methods for detecting a proteincapable of binding to porous hydroxyapatite. The hydroxyapatite may becomprised in enamel or dentine. Moreover, whereas existing productsstain dentine (but do not detect porous dental hydroxyapatite), for thefirst time disclosed herein is a product that detects defects in enamel,specifically by detecting porous dental hydroxyapatite.

The protein capable of binding to porous hydroxyapatite may be a humanprotein. For example, the protein may be selected from the group: Serumalbumin (P02768); Complement C3 beta chain (P01024); Alpha-1-antitrypsin(P01009); Protein S100-A9 (P06702); Lactotransferrin (P02788); Leukocyteelastase inhibitor (P30740); Antithrombin-III (P01008); Hemoglobinsubunit alpha (P69905); Hemoglobin subunit beta (P68871); Hemoglobinsubunit delta (P02042); Prolactin-inducible protein (P12273); Alphaamylase 1 (P04745); Ig kappa chain V-III region SIE (P01620); Ig alpha-2chain C region (P01877); Uncharacterized protein c6 or 158 (Q6P5S2);Serpin B3 (P29508), where the term in parentheses indicates the uniqueSwissProt accession identifier (as listed in Table 1, SEQ ID NOs: 1 to16 and FIGS. 6 to 21, respectively). In some embodiments, the proteinmay be an Amelogenin. The Amelogenin may be human. For example,amelogenin may be the X isoform of Human Amelogenin, (SEQ ID NO: 17,FIG. 22; SwissProt accession Q99217) or amelogenin may be the Y isoformof Human Amelogenin, (SEQ ID NO: 18, FIG. 23; SwissProt accessionQ99218). Alternatively, the protein may be from a subject other than ahuman, for example, an animal such as a primate, a horse, cow, sheep,goat, dog or cat.

In some embodiments of the first to fourth and seventh to ninth aspects,the protein may be albumin, hemoglobin or a subunit thereof, oramelogenin.

In one embodiment of the fourth aspect, the method comprises detectingthe protein which is other than amelogenin and detecting amelogenin,wherein presence of the protein and absence of amelogenin is indicativeof MIH, and presence of amelogenin is indicative of hypomaturationdefects including types or amelogenesis imperfecta or dental fluorosis.The protein which is “other than amelogenin” is any one selected from:Serum albumin; Complement C3 beta chain; Alpha-1-antitrypsin; ProteinS100-A9; Lactotransferrin; Leukocyte elastase inhibitor;Antithrombin-III; Hemoglobin subunit alpha; Hemoglobin subunit beta;Hemoglobin subunit delta; Prolactin-inducible protein; Alpha amylase 1;Ig kappa chain V-III region SIE; Ig alpha-2 chain C region;Uncharacterized protein c6orf58; and Serpin B3.

As used herein, “porous” or “porosity” refers to dental hydroxyapatitethat is either hypomineralised or demineralised. Increased “porosity” isdue to reduction in extent of mineral density, leading to increasedspace between mineral crystals.

“Hypomineralisation” as used herein, refers to incomplete development ofdental enamel, resulting in decreased mineral density (increased enamelporosity) and mechanical strength. “Hypomineralisation” is caused by agenetic (e.g. amelogenesis imperfecta) or acquired (e.g. MIH, fluorosis)disruption of dental development. “Hypomineralisation” is distinct from“demineralisation”, which occurs in caries for example. In caries,developmentally normal (or abnormal) enamel is subsequentlydemineralised. “Demineralisation” is distinct from “hypomineralisation”,which refers to enamel that never achieved normal mineral content due todisrupted development.

As used herein, “remineralisation” refers to the return of minerals tothe molecular structure of the tooth. The predominant mineral of teethis hydroxyapatite. In some remineralisation processes, the hydroxylgroup is substituted for a fluoro group to produce fluoroapatite, whichis more acid-resistant than hydroxyapatite.

As used herein, “remedial mineralisation” refers to the use ofremineralisation therapies on DDD (i.e. porous hydroxyapatite caused byincomplete mineralisation). Use of the term “remineralisation” isinappropriate in the DDD context because the porous hydroxyapatite wasnot caused by demineralisation.

As used herein, “caries” or “tooth decay” refers to reduction or loss oftooth enamel and dentine due to acid, particularly acid produced byinfecting bacteria. “Caries” is defined by the process ofdemineralisation, and may be corrected using remineralisation methods ifcaught early.

As used herein, a “condition involving porous dental hydroxyapatite”includes dental caries, Molar/Incisor Hypomineralisation (MIH),amelogenesis imperfecta, dental fluorosis and other DDD manifesting ashypomineralised enamel (i.e. diffuse or demarcated opacities).

As used herein, “Molar/Incisor Hypomineralisation” or “MIH” refers to aDDD that results in incompletely hardened (hypomineralised) enamel,usually on the occlusal or incisal third of first permanent molars andincisors, respectively.

MIH and fluorosis are both characterised by subsurface porosity, whereasactive caries can have a porous surface (inactive caries can form asealed surface due to remineralisation).

As used herein, “exposed” enamel refers to sub-surface tissue that hasbeen revealed due to loss of its protective surface layer. “Exposed”enamel may be normal or porous; there are many instances of surfacebreakdown on teeth that are not affected by MIH, or any other conditionfor that matter (e.g. otherwise normal teeth can fracture upon biting ahard object).

As used herein, “binds”, “binding” or “bound” refers to a chemicalinteraction between a protein and hydroxyapatite that arrests theprotein in relation to the hydroxyapatite. The interaction may be ionic,covalent, non-covalent, polar or non-polar.

As used herein, the term “detector” refers to any chemical, biochemicalor biological substance that interacts specifically with a proteindisclosed herein and generates an effect in response to the interaction.For example, the response may be visualisation of a coloured reporter,and thus visualisation of the protein. A “detector” may comprise a“reporter” or an antibody.

The term “detect” or “detecting” refers to identifying the response fromthe detector.

In one embodiment of the kit of the first aspect, the detector comprisesa coloured reporter. In the probe of the second aspect, the detector isa reporter. In one embodiment of the probe, the reporter comprises acoloured reporter. When the detector comprises a coloured reporter,detecting the coloured reporter would involve visualising the colouredreporter and therefore the protein of interest. Alternatively, areporter may be radio-opaque.

As used herein, the term “probe” refers to an agent such as a proteindisclosed herein that can infiltrate porous enamel and that canspecifically and tightly bind to hydroxyapatite and upon binding enablesuch binding to be detected. In other words, the probe comprises aspecific “hydroxyapatite-targeting” molecule. A “probe” comprises aprotein as disclosed herein and a reporter. According to thisdisclosure, a “probe” may not be an antibody.

Similarly, the term “specific” or “specifically” refers to binding whereone substance binds to a particular second substance withoutsubstantially binding to any other substance. Such binding is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target, for example, an excess of non-labeled target. Inthis case, specific binding is indicated if the binding of the labeledtarget to a probe is competitively inhibited by excess unlabeled target.As used herein, “specific” or “specifically” binding may refer to (i)the protein binding specifically to hydroxyapatite, (ii) the detectorspecifically binding to the protein, or (iii) the reporter specificallybinding to the detector or protein.

In particular, specific binding refers to a substance having a K_(d) atleast 2-fold less than that of a non-specific target, for example, asubstance having a K_(d) at least 4-fold, 6-fold, 8-fold, 10-fold, ormore than 10-fold less than that of a non-specific target.Alternatively, specific binding can be expressed as a molecule having aK_(d) for the target of at most about 10⁻⁴ M, for example, about 10⁻⁵ M,about 10⁻⁶ M, about 10⁻⁷ M, about 10⁻⁸ M, about 10⁻⁹ M, about 10⁻¹⁰ M,about 10⁻¹¹ M, about 10⁻¹² M, or less.

In one embodiment of the kit of the first aspect, the detector comprisesa reporter.

When used in situ, the detector or probe is non-toxic to the subject.

As used herein, a “reporter” refers to any chemical, biochemical orbiological substance that generates a detectable effect. The “reporter”may specifically bind to or be linked to the detector or protein. Thereporter may comprise biotin or streptavidin for use in a high affinity,non-covalent biotin-streptavidin bond. The reporter may exploit anotherhigh affinity, non-covalent bond.

In one embodiment of the kit of the first aspect or the probe of thesecond aspect, the reporter may be a coloured reporter. In otherembodiments of the first or second aspect, the reporter may be apigment, or a luminescent (including fluorescent or phosphorescent),radioactive, chemiluminescent substance, enzyme, or x-ray contrastmolecule. A reporter comprising an X-ray contrast molecule (e.g.5-amino-2,4,6-triiodoisophthalic acid; ³I) may be of use for sensitivelydetecting early-stage interproximal caries (a major challenge forcurrent methods) using existing clinical radiographic equipment.

As used herein, the term “coloured reporter” refers to any colouredsubstance that absorbs some wavelengths of visible light preferentially.

Thus, when the reporter is a coloured reporter, the detectable effect isvisualisation of a colour.

The coloured reporter may be any coloured substance that is amenable tolinking, coupling or conjugating to the protein, whilst maintaining itscharacteristic as a coloured reporter. In one embodiment of the kit ofthe first aspect or probe of the second aspect, the coloured reporter isamido black. In one example, the probe comprises the protein (i.e. ahydroxyapatite-binding-protein) linked or coupled to a colouredreporter. Any protein from Table 1 may be linked or coupled to acoloured reporter and function to target the coloured reporter to poroushydroxyapatite. In one example, the protein is hemoglobin. Thus, in oneexample, the probe comprises hemoglobin linked to amido black.

A probe comprising a protein as disclosed herein, e.g. haemoglobin, isadsorbed cumulatively to porous dental hydroxyapatite. The probe willcompetitively bind to hydroxyapatite in the presence of other proteinsbecause it is able to displace any species possessing lower affinity forhydroxyapatite. Such a probe may comprise amido black or ³I.

The coloured reporter may be selected, based on desired features thatwould be known to a person skilled in the art, from the group: Acetylyellow (Fast yellow); Acid black 1 (Amido black 10B); Acid blue 22(Water blue I); Acid blue 93 (Methyl blue); Acid fuchsin (Acid fuchsin);Acid green (Light green SF yellowish); Acid green 1 (Naphthol green B);Acid green 5 (Light green SF yellowish); Acid magenta (Acid fuchsin);Acid orange 10 (Orange G); Acid red 4 (Azo-eosin); Acid red 26 (Xylidineponceau); Acid red 29 (Chromotrope 2R); Acid red 44 (Ponceau 6R); Acidred 51 (Erythrosin B); Acid red 52 (Lissamine rhodamine B); Acid red 66(Biebrich scarlet); Acid red 73 (bloodstain scarlet); Acid red 87 (EosinY ws); Acid red 91 (Eosin B); Acid red 92 (Phloxine B); Acid red 94(Rose bengal); Acid red 101 (Azocarmine G); Acid red 103 (Azocarmine B);Acid roseine (Acid fuchsin); Acid rubin (Acid fuchsin); Acid violet 19(Acid fuchsin); Acid yellow 1 (Naphthol yellow S); Acid yellow 7(Lissamine flavine FF); Acid yellow 9 (Fast yellow); Acid yellow 23(Tartrazine); Acid yellow 24 (Martius yellow); Acid yellow 36 (Metanilyellow); Acid yellow 73 (Fluorescein); Acid yellow 85 (Coomassie fastyellow G); Acid yellow S (Naphthol yellow S); Acid yellow T(Tartrazine); Acridine orange (Acridine orange); Acridine red (Acridinered); Acriflavine (Acriflavine); Alcian blue (Aldan blue 8GX); Alcianyellow (Alcian yellow); Alcohol soluble eosin (Ethyl eosin); Alizarin(Alizarin); Alizarin blue (Alizarin blue); Alizarin blue 2RC (Anthraceneblue SWR); Alizarin carmine (Alizarin red S); Alizarin cyanin BBS(Alizarin cyanin BBS); Alizarol cyanin R (Chromoxane cyanin R); Alizarinred S (Alizarin red S); Alizarin purpurin (Purpurin); Alkali blue 4B, 5B(Alkali blue 5B); Aluminon (Chrome violet CG); Amido black 10B (Amidoblack 10B); Amidonaphthol red (Azophloxine); Amidoschwarz (Amido black10B); Aniline blue WS (Aniline blue WS); Aniline purple (Mauveine);Anthracene blue SWR (Anthracene blue SWR); Anthracene blue SWX (Alizarincyanin BBS); Auramine O (Auramine O); Azo-eosin (Azo-eosin); AzocarmineB (Azocarmine B); Azocarmine G (Azocarmine Azoeosin G (Azo-eosin); Azoicdiazo 5 (Fast red B); Azoic diazo 48 (Fast blue B); Azophloxine(Azophloxine); Azovan blue (Evans blue); Azure A (Azure A); Azure B(Azure B); Azure C (Azure C); Basic blue 8 (Victoria blue 4R); Basicblue 9 (Methylene blue); Basic blue 12 (Nile blue A); Basic blue 15(Night blue); Basic blue 17 (Toluidine blue O); Basic blue 20 (Methylgreen); Basic blue 26 (Victoria blue B); Basic brown 1 (Bismarck brownY); Basic fuchsin (Basic fuchsin); Basic green 4 (Malachite green);Basic green 5 (Methylene green); Basic orange 14 (Acridine orange);Basic red 2 (Safranin O); Basic red 5 (Neutral red); Basic red 9(Pararosanilin); Basic violet 2 (New fuchsin); Basic violet 3 (Crystalviolet); Basic violet 4 (Ethyl violet); Basic violet 10 (Rhodamine B);Basic violet 14 (Rosanilin); Basic yellow 1 (Thioflavine T); Basicyellow 2 (Auramine O); Biebrich scarlet (Biebrich scarlet); Biebrichscarlet R (Sudan IV); Bismarck brown Y (Bismarck brown Y); Blauschwarz(Naphalene blue black CS); Brazilein (Brazilein); Brazilin (Brazilin);Brilliant crocein (Woodstain scarlet); Brilliant crystal scarlet 6R(Ponceau 6R); Brilliant green (Brilliant green); Calcium red (Nuclearfast red); Carmine (Carmine); Carminic acid (Carmine); Carmoisine 6R(Chromotrope 2R); Celestine blue B (Celestine blue B); China blue(Aniline blue); Chlorantine fast red 5B (Sirius red 4B); Chicago blue 4B(Pontamine sky blue 5B); Chrome fast yellow 8GL (Chrome fast yellow8GL); Chrome luxine yellow 8G (Chrome fast yellow 8GL); Chrome violet CG(Chrome violet CG); Chromotrope 2R (Chromotrope 2R); Chromoxane cyanin R(Chromoxane cyanin R); Cochineal (Carmine); Coelestine blue (Celestineblue B); Congo corinth (Congo Corinth); Congo red (Congo red); Coomassiefast yellow G (Coomassie fast yellow G); Cotton blue (Methyl blue);Cotton red (Congo red); Croceine scarlet (Biebrich scarlet); Croceinscarlet 3B (Woodstain scarlet); Crocein scarlet MOO (Woodstain scarlet);Crocin (Saffron); Crystal ponceau 6R (Ponceau 6R); Crystal scarlet(Ponceau 6R); Crystal violet (Crystal violet); Dahlia (Hoffman'sviolet); Diamond green B (Malachite green); Direct blue 14 (Trypanblue); Direct blue 58 (Evans blue); Direct red (Congo red); Direct red10 (Congo corinth); Direct red 28 (Congo red); Direct red 80 (Sirius redF3B); Direct red 81 (Sirius red 4B); Direct yellow 7 (Thioflavine S);Direct yellow 11 (Sun yellow); Durazol blue 4R (Durazol blue 4R);Durazol blue 8G (Durazol blue 8G); Eosin B (Eosin B); Eosin Bluish(Eosin B); Eosin (Eosin Y ws); Eosin Y (Eosin Y ws); Eosin yellowish(Eosin Y ws); Eosinol (Eosinol); Erie garnet B (Congo corinth);Eriochrome cyanin R (Chromoxane cyanin R); Erythrosin B (Erythrosin B);Ethyl eosin (Ethyl eosin); Ethyl green (Ethyl green); Ethyl violet(Ethyl violet); Evans blue (Evans blue); Fast blue B (Fast blue B); Fastgreen FCF (Fast green FCF); Fast red B (Fast red B); Fast yellow (Fastyellow); Fast yellow extra (Fast yellow); Fast yellow G (Fast yellow);Fat black HB (Sudan black B); Fluorescein (Fluorescein); Food green 3(Fast green FCF); Gallein (Gallein); Gallamine blue (Gallamine blue);Gallocyanin (Gallocyanin); Gentian violet (Methyl violet 2B); Guineegreen (Guinee green B); Haematein (Hematein); Haematine (Hematein);Haematoxylin (Hematoxylin); Helio fast rubin BBL (Nuclear fast red);Helvetia blue (Methyl blue); Hematein (Hematein); Hematine (Hematein);Hematoxylin (Hematoxylin); Hoffman's violet (Hoffman's violet);Hydrazine yellow (Tartrazine); Indigo carmine (Indigo carmine); Imperialred (Eosin B); Ingrain blue 1 (Alcian blue 8GX); Ingrain yellow 1(Alcian yellow); INT (Iodonitrotetrazolium); Iodine green (Iodinegreen); Kermes (Kermes); Kermesic acid (Kermes); Kemechtrot (Nuclearfast red); Kiton rhodamine B (Lissamine rhodamine B); Lac (Laccaicacid); Laccaic acid (Laccaic acid); Lauth's violet (Thionin); Lightgreen (Light green SF yellowish); Lissamine fast yellow (Lissamine fastyellow); Lissamine flavine FF (Lissamine flavine FF); Lissamine green SF(Light green SF yellowish); Lissamine rhodamine B (Lissamine rhodamineB); Luxine pure yellow 6G (Chrome fast yellow 8GL); Luxol fast blue(Luxol fast blue MBS); Magenta 0 (Pararosanilin); Magenta I (Rosanilin);Magenta II (Magenta II); Magenta III (New fuchsin); Malachite green(Malachite green); Manchester brown (Bismarck brown Y); Martius yellow(Martius yellow); Mauve (Mauveine); Mauveine (Mauveine); Merbromin(Mercurochrome 220); Mercurochrome (Mercurochrome 220); Metanil yellow(Metanil yellow); Methyl blue (Methyl blue); Methyl green (Methylgreen); Methyl violet (Methyl violet 2B); Methyl violet 2B (Methylviolet 2B); Methyl violet 10B (Crystal violet); Methylene azure A (AzureA); Methylene azure B (Azure B); Methylene azure C (Azure C); Methyleneblue (Methylene blue); Methylene green (Methylene green); Milling yellow3G (Milling yellow 3G); Mordant blue 3 (Chromoxane cyanin R); Mordantblue 10 (Gallocyanin); Mordant blue 14 (Celestine blue B); Mordant blue23 (Alizarin cyanin BBS); Mordant blue 32 (Anthracene blue SWR); Mordantblue 45 (Gallamine blue); Mordant red 3 (Alizarin red S); Mordant red 11(Alizarin); Mordant violet 25 (Gallein); Mordant violet 39 (Chromeviolet CG); Mordant yellow 33 (Chrome fast yellow 8GL); Naphthalene blueblack (Naphalene blue black CS); Naphthol blue black (Amido black 10B);Naphthol green B (Naphthol green B); Naphthol yellow S (Naphthol yellowS); Natural black 1 (Hematein); Natural red (Purpurin); Natural red 3(Kermes); Natural red 4 (Carmine); Natural red 8 (Purpurin); Natural red16 (Purpurin); Natural red 24 (Brazilin); Natural red 25 (Laccaic acid);Natural red 28 (Orcein); Natural yellow 6 (Saffron); NBT (Nitro bluetetrazolium); Neutral red (Neutral red); New fuchsin (New fuchsin);Niagara blue 3B (Trypan blue); Night blue (Night blue); Nile blue (Nileblue A); Nile blue A (Nile blue A); Nile blue sulphate (Nile blue A);Nile red (Nile red); Nitro BT (Nitro blue tetrazolium); Nitro bluetetrazolium (Nitro blue tetrazolium); Nuclear fast red (Nuclear fastred); Oil red O (Oil red O); Orange G (Orange G); Orcein (Orcein);Pararosanilin (Pararosanilin); Perkin's violet (Mauveine); Phloxine B(Phloxine B); Picric acid (Picric acid); Ponceau 2R (Xylidine ponceau);Ponceau 6R (Ponceau 6R); Ponceau B (Biebrich scarlet); Ponceau deXylidine (Xylidine ponceau); Ponceau S (Ponceau S); Pontamine sky blue5B (Pontamine sky blue 5B); Primula (Hoffman's violet); Primuline(Primuline); Purpurin (Purpurin); Pyronin B (Pyronin B); Pyronin G(Pyronin Y); Pyronin Y (Pyronin Y); Rhodamine B (Rhodamine B); Rosanilin(Rosanilin); Rose bengal (Rose bengal); Saffron (Saffron); Safranin O(Safranin O); Scarlet R (Sudan IV); Scarlet red (Sudan IV); Scharlach R(Sudan IV); Shellac (Laccaic acid); Sirius red F3B (Sirius red F3B);Sirius red 4B (Sirius red 4B); Sirius supra blue F3R (Durazol blue 4R);Solochrome cyanin R (Chromoxane cyanin R); Soluble blue (Aniline blue);Solvent black 3 (Sudan black B); Solvent blue 38 (Luxol fast blue MBS);Solvent red 23 (Sudan III); Solvent red 24 (Sudan IV); Solvent red 27(Oil red O); Solvent red 45 (Ethyl eosin); Solvent yellow 94(Fluorescein); Spirit soluble eosin (Ethyl eosin); Sudan III (SudanIII); Sudan IV (Sudan IV); Sudan black B (Sudan black B); Sudan red BK(Sudan III); Sulfur yellow S (Naphthol yellow S); Sulpho rhodamine B(Lissamine rhodamine B); Sun yellow (Sun yellow); Swiss blue (Methyleneblue); Tartrazine (Tartrazine); Thioflavine S (Thioflavine 5);Thioflavine T (Thioflavine T); Thionin (Thionin); Toluidine blue(Toluidine blue O); Toluyline red (Neutral red); Tropaeolin G (Metanilyellow); Trypaflavine (Acriflavine); Trypan blue (Trypan blue); Uranin(Fluorescein); Victoria blue 4R (Victoria blue 4R); Victoria blue B(Victoria blue B); Victoria blue R (Victoria blue R); Victoria green B(Malachite green); Water blue I (Water blue I); Water soluble eosin(Eosin Y ws); Woodstain scarlet (Woodstain scarlet); Xylene red B(Lissamine rhodamine B); Xylidine ponceau (Xylidine ponceau); andYellowish eosin (Eosin Y ws). The desired features to be considered bythe skilled addressee include compatibility with a protein and/or alinker to be used according to this disclosure, non-toxicity, andmaintenance of protein binding to porous dental hydroxyapatite, forexample.

In alternative embodiments of the first to fifth or seventh to ninthaspects, the protein is not listed in the examples of Table 1, but isknown to the skilled addressee to bind to hydroxyapatite, for exampleosteocalcin or decorin. Use of the leucine-rich repeat domains 4-5 fromdecorin may provide a specific targeting mechanism for poroushydroxyapatite in dentine.

Alternatively, in embodiments of the first to fifth or seventh to ninthaspects, the protein may be a peptide or protein fragment, provided thatthe peptide or protein fragment retains its ability to bind to porousdental hydroxyapatite.

Alternatively, the skilled addressee will be aware of small molecules(or polymers thereof), for example tetracycline or amino bisphosphonate,that can bind to hydroxyapatite, which may be of more use in terms ofability to penetrate micro-porous regions, and in terms of stability(e.g. product shelf-life). Amino-bisphosphonate may produce a compoundwith qualities suited to detecting and delineating caries (small,high-affinity probe for penetrating porous enamel surface and stronglybinding to demineralised enamel).

In some embodiments of the kit of the first aspect or probe of thesecond aspect, the detector or probe further comprises a linker linkingthe reporter and the detector or protein. The linker may be aheterobifunctional cross-linker. For example, the heterobifunctionallinker may be succinimidyl 4-[N-maleimidomethyl]cyclohexanecarboxylicacid N-hydroxysuccinimide ester (SMCC). Other examples of linking agentsthat may be used in accordance with this disclosure includesuccinimidyl-6-[β-maleimidopropionamido]hexanoate (SMPH),N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA), andN,N-dicyclohexylcarbodiimide (DCC).

Other types of molecules may be used as a linker. For example, highaffinity, non-covalent bonds such as biotin-streptavidin are alsocontemplated herein.

The skilled addressee will be aware of many cross-linking agents thatare available with various reactive chemistries and spacer-arm lengths,further increasing the flexibility of this approach.

In one embodiment of the kit of the first aspect or probe of the secondaspect, the reporter and the protein may be provided already linked.Alternatively, the reporter and protein may be provided separately forsubsequent linkage. The kit or probe may comprise a linker. The protein,reporter and linker of the kit or probe may be presented in any possiblecombination. For example, when the reporter and protein are linked via alinker, the probe may be “ready-to-use”, i.e. the three components maybe linked. Alternatively, the protein and the linker may be linked andprovided separately to the reporter. Alternatively, the linker and thereporter may be linked and provided separately to the protein.Alternatively, the protein, the reporter, and the linker may be providedas separate components. In one embodiment of the first aspect, the kitwill comprise the reporter and the linker, but not the protein.

In some embodiments of the kit of the first aspect, the detectorcomprises an antibody that specifically binds the protein. In anotherembodiment, the detector may comprise biotin or streptavidin for use ina high affinity, non-covalent biotin-streptavidin bond. The detector mayexploit another high affinity, non-covalent bond. For example, thedetector may comprise an antibody alternative, such as a peptide-basedprotein ligand. A peptide-based protein ligand known in the art is asynbody.

The term “antibody” is used in the broadest sense and specificallycovers, for example, polyclonal antibodies, monoclonal antibodies(including antagonist and neutralizing antibodies), antibodycompositions with polyepitopic specificity, single chain antibodies, andfragments of antibodies, provided that they exhibit the desiredbiological or immunological activity. The antibody may be a conjugatedantibody or any other type of antibody known to the person skilled inthe art.

The antibody may be detected by any method known to the person skilledin the art. The primary antibody may comprise a reporter. Alternatively,a secondary antibody targeting the primary antibody may comprise areporter.

The antibody may be any antibody known by the skilled addressee tospecifically bind to a protein selected from the group: Serum albumin;Complement C3 beta chain; Alpha-1-antitrypsin; Protein S100-A9;Lactotransferrin; Leukocyte elastase inhibitor; Antithrombin-III;Hemoglobin subunit alpha; Hemoglobin subunit beta; Hemoglobin subunitdelta; Prolactin-inducible protein; Alpha amylase 1; Ig kappa chainV-III region SIE; Ig alpha-2 chain C region; Uncharacterized proteinc6orf58; and Serpin B3. In one embodiment, the antibody may specificallybind to an amelogenin.

In one embodiment of the kit of the first aspect, an anti-serum albuminmonoclonal antibody may be selected from the group: AL-01; 1.B.731; 1A9;6B11; OCH1E5; 1C8; 1G2; 2B2; 2B3; 2B6; 14E7; 15C7; Alb1; and a mountmonoclonal IgG₁ antibody with product code sc-70340 (Santa CruzBiotechnology Inc). In one embodiment, an anti Complement C3 beta chainmonoclonal antibody may be clone 755. In another embodiment, ananti-human C3 monoclonal antibody that cross-reacts with Complement C3beta chain may be used and may be clone 11H9. In one embodiment, ananti-Alpha-1-antitrypsin monoclonal antibody may be selected from thegroup: 5B12; 703; 704; 8A0; B9; and G11. In one embodiment, ananti-Protein S100-A9 monoclonal antibody may be selected from the group:0.N.390A; 47-8D3; N0.134; N0.19; and S32.2. In one embodiment, ananti-Lactotransferrin monoclonal antibody may be selected from thegroup: 1C6; 2B8; B97; CLB-13.17; and 1A1. In one embodiment, ananti-Antithrombin-III monoclonal antibody may be 4B3 or BDI205. In oneembodiment, an anti-Hemoglobin subunit alpha antibody may be a goatpolyclonal IgG antibody with product code sc-70340 (Santa CruzBiotechnology Inc). In one embodiment, an anti-Hemoglobin subunit betaantibody may be a mouse monoclonal IgG₁ antibody with product codesc-21757 (Santa Cruz Biotechnology Inc). In one embodiment, an anti-Igalpha-2 chain C region monoclonal antibody may be clone 14AS (alsoreferred to as anti-human IgA2). In one embodiment, an anti-Amelogenin Xantibody may be a rabbit polyclonal IgG antibody with product codesc-32892 (Santa Cruz Biotechnology Inc). The skilled addressee willappreciate that other suitable antibodies are available.

In one embodiment of the kit, probe or method of the first, second orfourth aspects, a first protein is selected from the group: Serumalbumin; Complement C3 beta chain; Alpha-1-antitrypsin; Protein S100-A9;Lactotransferrin; Leukocyte elastase inhibitor; Antithrombin-III;Hemoglobin subunit alpha; Hemoglobin subunit beta; Hemoglobin subunitdelta; Prolactin-inducible protein; Alpha amylase 1; Ig kappa chainV-III region SIE; Ig alpha-2 chain C region; Uncharacterized proteinc6orf58; and Serpin B3 may be detected, and a second protein may bedetected, wherein the second protein is amelogenin. It follows that akit of the first aspect may also comprise a second detector that detectsamelogenin. The second detector may be an anti-amelogenin antibody.

Alternatively, detecting may comprise immunodetection, chromatography,electrophoresis, mass spectrometry, or microscopy. Immunodetection maycomprise enzyme-linked immunosorbent assay (ELISA), Western Blot, dotblot, slot blot, or flow cytometry, for example. Microscopy may compriseconfocal laser, fluorescence or electron microscopy, for example.

The detector or probe may be applied in different ways, for example in aliquid, gel, capsule, tablet, aqueous solution, aqueous or oilysuspension, lozenge, troche, powder, granule, emulsion, syrup or elixir.

In one embodiment of the kit of the first aspect or probe of the secondaspect, the detector or probe comprises a solvent in which the detectoror probe is dissolved, suspended or emulsified. The solvent may be onethat is used generally in medicine or industry or similar. Examplesinclude water, ethanol, n-propanol, 2-butyl alcohol, isobutyl alcohol,n-amyl alcohol, isoamyl alcohol, ethylene glycol, 2-methoxyethanol,diethylene glycol, triethylene glycol, tetraethylene glycol,polyethylene glycol, propylene glycol, dipropylene glycol, polypropyleneglycol, trimethylene glycol, 1,2-butanediol, 1,3-butanediol,2,3-butanediol, 1,4-butanediol, 1,5-pentanediol, ethylene glycolmonomethyl ether, ethylene glycol monomethyl ether acetate, ethyleneglycol monoethyl ether, ethylene glycol diethylether,ethyleneglycolmonoethyletheracetate, ethylene glycol isopropyl ether,ethylene glycol monobutyl ether, ethylene glycol dibutyl ether, ethyleneglycol monoacetate, ethylene glycol diacetate, diethylene glycolmonomethyl ether, diethylene glycol monoethyl ether, diethylene glycolmonoethyl ether acetate, diethylene glycol monobutyl ether, diethyleneglycolmonobutyl ether acetate, diethylene glycol dimethyl ether,diethylene glycol methylethyl ether, diethylene glycol diethyl ether,diethylene glycol acetate, triethylene glycol monomethyl ether,triethylene glycol monoethyl ether, propylene glycol monomethyl ether,propylene glycol monoethyl ether, dipropylene glycol monomethyl ether,dipropylene glycol monoethyl ether, ee tripropylene glycol monomethylether, glycerin, tetrahydrofuran, dimethylformamide, dioxane, acetone,and dimethoxyethane.

In some embodiments, the solvent comprises water, ethanol, glycerin,isobutyl alcohol, ethylene glycol, diethylene glycol, triethyleneglycol, acetone, or propylene glycol, which are compatible with humans.

One solvent may be used singly or two or more solvents may be used inadmixture.

The detector may be compounded with a thickener to increase itsviscosity to about 50 to about 2 000 mPa·s, for example 100, 200, 300,400, 500, 750, 1000, 1250, 1500, or 1750 mPa s (at 25° C.), therebyforming a gel. In gel form, applying the detector with a toothbrushenables simultaneous cleaning of the tooth and application of thedetector.

Examples of thickeners that may be used include: synthetic additivessuch as sodium alginate, propylene glycol alginate, sodium carboxymethylcellulose, calcium carboxymethyl cellulose, sodium carboxymethyl starch,sodium starch phosphate, sodium polyacrylate, methyl cellulose,hydroxypropyl cellulose, and polyvinylpyrrolidone; natural thickenerssuch as cyamoposis gum, Carob bean gum, Tara gum, Tamarind seed gum, gumarabic, tragacanth gum, Karaya gum, alginic acid, carrageenan, xanthangum, gellan gum, curdlan, chitin, chitosan, and chitosamine; andinorganic thickeners such as calcium carbonate, calcium silicate, silicapowder, amorphous hydrous silicate, and hydrophobic silica.

In order to obtain viscosity in the range of about 50 to about 2 000mPa·s, the compounding amount of the thickener varies depending on thekind of the thickener. For example, when sodium carboxymethyl cellulosehaving a large thickening effect, the compounding amount may be about0.5 to 4% by weight, and when methyl cellulose, the compounding amountmay be about 10 to 30% by weight.

Furthermore, the detector or probe may comprise additives such assweeteners, flavours, and preservatives. Suitable sweeteners includesucrose, lactose, glucose, aspartame or saccharin. Suitable flavouringagents include peppermint oil, oil of wintergreen, cherry, orange orraspberry flavouring. Suitable preservatives include sodium benzoate,vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propylparaben or sodium bisulphite. Suitable lubricants include magnesiumstearate, stearic acid, sodium oleate, sodium chloride or talc. Suitabledisintegrating agents include corn starch, methylcellulose,polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Atablet may contain the detector in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets.

In another embodiment, the kit of the first aspect further comprises oneor more washing solutions.

The kit of the seventh or ninth aspect comprises one or more washingsolutions.

A washing solution of the kit of the first, seventh or ninth aspect maycomprise a solution to remove any protein not specifically bound toporous hydroxyapatite, i.e. non-desorbing. For example, a washingsolution that does not desorb a protein bound to hydroxyapatite may bewater, saline, Tris buffer, or mild detergent etc. As the oral cavitycontains abundant proteins including many of the proteins that bindhydroxyapatite, a washing solution allows protein not specifically boundto hydroxyapatite to be removed from the tooth or sample thereof priorto application of the detector.

In other embodiments of the kit of the first, seventh or ninth aspect,the washing solution comprises magnesium ions (Mg²⁺),dihydrogenphosphate ions (H₂PO₄ ⁻), hydrogenphosphate ions (HPO₄ ²⁻), orphosphate ions (PO₄ ³⁻) (collectively “PO₄”), or may comprise aplurality of washing solutions that may each comprise magnesium ions(Mg²⁺), dihydrogenphosphate ions (H₂PO₄ ⁻), hydrogenphosphate ions (HPO₄²⁻), or phosphate ions (PO₄ ³⁻), administrable sequentially. Any solublemagnesium salt may be used and any soluble dihydrogenphosphate,hydrogenphosphate ions (HPO₄ ²⁻), or phosphate salt may be used,provided that it is non-toxic if applied in situ. In one embodiment, thewashing solution comprises magnesium chloride or sodiumdihydrogenphosphate. The skilled addressee will appreciate that otherwashing solutions capable of desorbing protein from hydroxyapatite areavailable.

A washing solution may comprise hypochlorous acid (HOCl), hypochlorite(NaOCl) or calcium hypochlorite (Ca(OCl)₂) (collectively “bleach”).

A washing solution may be provided ready to use. Alternatively, thewashing solution may be provided as a concentrate to prepare the washingsolution upon dilution with water. Alternatively, the washing solutionmay be provided as one or more dry components to prepare the washingsolution upon admixture with water.

The washing solution may comprise less than 1 mM, about 1 mM, about 2mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8mM, about 9 mM, about 10 mM or more than 10 mM magnesium ions. Thewashing solution may comprise less than 0.1 M, about 0.1 M, about 0.5 M,about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1 M, about 1.1M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 2 M, about10 M or more than 10 M magnesium ions. The washing solution may compriseless than 0.04 M, about 0.04 M, about 0.08 M, about 0.09 M, about 0.1 M,about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about0.7 M, about 0.8 M, about 0.9 M, about 1 M, about 1.5 M, about 2 M,about 4 M, or more than 4 M dihydrogenphosphate, hydrogenphosphate orphosphate ions.

The washing solution may comprise about 10% bleach (about 0.4% ⁻OCl),neat or undiluted bleach (about 4% ⁻OCl), or may comprise about 20%(about 0.8% ⁻OCl), about 30% (about 1.2% ⁻OCl), about 40% (about 1.6%⁻OCl), about 50% (about 2.0% ⁻OCl), about 60% (about 2.4% ⁻OCl), about70% (about 2.8% ⁻OCl), about 80% (about 3.2% ⁻OCl), about 90% (about3.6% ⁻OCl) or about 95% bleach (about 3.8% ⁻OCl).

While not wishing to be bound to any particular theory, it is thoughtthat providing a plurality of washing solutions with a step-wiseconcentration gradient of magnesium and/or phosphate removes moreproteins than a single concentration magnesium solution. It is thoughtthat bleach (⁻OCl) non-specifically strips bound proteins fromhydroxyapatite.

Thus, in one embodiment of the kit of the first, seventh or ninthaspect, the one or more washing solutions, or plurality of washingsolutions, may comprise a solution of about 5 mM magnesium chloride, asolution of about 1 M magnesium chloride, and/or a solution of about 0.4M sodium dihydrogenphosphate.

Where one or more (a plurality) of washing solutions is applied, thewashing solutions may be applied in any order. Alternatively, where oneor more (a plurality) of washing solutions is applied, the washingsolutions may be applied sequentially in the order of low magnesiumconcentration (e.g. 5 mM), high magnesium concentration (e.g. 1 M),dihydrogenphosphate (e.g. 0.4 M; or hydrogenphosphate or phosphate).Alternatively, a washing solution may comprise in combination magnesiumand phosphate, for example, about 1 M magnesium concentration and about0.4 M dihydrogenphosphate, hydrogenphosphate or phosphate.

Washing may occur before detecting, or after detecting, or before andafter detecting.

As used herein, “removes” or “removing” refers to a reduction in theconcentration of protein bound to hydroxyapatite.

As used herein, a “sample” is a portion or part of the tooth to be usedfor detection or diagnosis of porous hydroxyapatite. A “control sample”is a portion or part of the tooth known to be healthy and free of poroushydroxyapatite and is used for reference purposes when detecting ordiagnosing porosity in test hydroxyapatite. A “sample” may be obtainedby wiping, swabbing, scraping, chipping, drilling or similar. A samplermay be adapted for obtaining a sample by swabbing, wiping or any othermethod of collection known to the skilled addressee.

In one embodiment of the method of the fourth to sixth aspect, the toothis first cleaned by brushing or other means. The tooth may be dried.

In one embodiment of the kit of the first aspect, probe of the secondaspect, or method of the fourth to sixth aspect, the detector is appliedusing a brush, toothbrush, a cotton swab, a cotton ball or by droppingfrom a nozzle-equipped container.

As used herein, “applied”, “applying” or “application” has its ordinarymeaning of bringing into contact the detector or probe or washingsolution and the tooth or sample thereof, or bringing into contact thereporter and the detector or protein.

After application of the detector or probe, the detector or probe isincubated on the tooth for a period of time sufficient for binding ofthe detector to the protein or for binding of the probe to thehydroxyapatite. The incubation period may be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 s. Alternatively, theincubation time may be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 min.Alternatively, the incubation time may be more than 5 min, such as 10,15 or 20 min. After incubation, excess detector or probe may bedisgorged from the mouth with or without washing using water or awashing solution.

The method or use may be performed in or on the tooth in situ in asubject. Alternatively, the method or use may be performed in or on thetooth or a sample of the tooth after removal from a subject.

The subject includes a mammal. The mammal may be a human. The human maybe any age. The human may be under about 12 years of age. The human maybe about 2 to about 12 years of age, about 4 to about 10 years of age,or about 6 to about 10 years of age. Alternatively, the subject may be12 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80to 90, or 90 to 100 years of age.

The subject may develop porous hydroxyapatite after normalhydroxyapatite and normal enamel has developed, or the subject may haveporous hydroxyapatite throughout development.

Alternatively, the subject may be a domestic, zoo, or companion animal.While it is particularly contemplated that the methods and uses hereinare suitable for humans, they are also applicable to primates, companionanimals such as dogs and cats, domestic animals such as horses, cattle,sheep and goats, zoo animals such as felids, canids, bovids, andungulates, or laboratory animals such as lagomorphs and rodents. Asubject may be afflicted with a dental disorder, or may not be afflictedwith a dental disorder (i.e., free of detectable disease).

The diagnostic power of the kits or methods disclosed herein is based onconditions of porous hydroxyapatite (DDD and caries) havingdistinguishable protein profiles (e.g. MIH: abundant proteins from Table1, little or no amelogenin; mature fluorosis: trace amounts of albuminand amelogenin; hypomaturation amelogenesis imperfecta: abundantamelogenin). Different defects may require different wash proceduresbefore remedial mineralisation, or different restoration methods andmaterials (or influence the choice thereof). Protein concentration intest enamel of a tooth or sample thereof may be assessed by variousmeans, and the condition involving porous hydroxyapatite can bediagnosed based on the identity of proteins with elevated abundancerelative to control.

A further application of the present disclosure is to categorise the MIHlesion sub-type (e.g. as intact or broken), which may impact the type oftreatment required (e.g. different protein compositions may needdifferent wash procedures before remedial mineralisation).

As used herein, “intact” has its ordinary meaning of undisrupted,uninjured or unaltered and is used in relation to the surface of toothenamel. “Intact” here refers to a lesion covered with a shell of harderenamel at the tooth surface, and is referred to as a subsurface lesion,indicating a stratified structure.

In contrast, “broken” here refers to an MIH lesion whose hard enamelshell either has become disrupted due to mechanical forces, or was notpresent initially (perhaps lost during tooth eruption, or not producedduring development).

As used herein, “permeable surface” refers to intact or broken enamelthat allows access of oral fluid or any other solution (and associatedcomponents including proteins) into subsurface regions. Conversely, an“impermeable surface” refers to intact or broken enamel that blocks suchaccess.

MIH lesions comprising intact enamel, despite comprising poroushydroxyapatite, may or may not present porous dental hydroxyapatiteamenable to detection (i.e. may have a permeable or impermeablesurface). Therefore, in some embodiments of the first, seventh or ninthaspects, a kit may comprise a permeabilising agent, or a method of thefourth to sixth aspects (e.g. mechanical permeabilisation) may comprisepermeabilising the tooth or a sample of the tooth. Such an agent ormethod will be used in pre-treating a lesion that has an impermeablesurface. Alternatively, such an agent may be used to access a lesionpreviously subject to remineralisation or remedial mineralisation.

As used herein, “permeabilise” or “permeabilising” refers to openingpores in impermeable enamel of sufficient dimension to enable thedetector and/or the washing solution access to the poroushydroxyapatite, and/or to enable protein removal.

The permeabilisation agent is a formulation capable of permeabilisingthe surface layer of enamel (e.g. it may comprise an acid or some otheragent known by the skilled addressee to permeabilise enamel). Thepermeabilisation agent may be in the form of a solution or a gel, forexample.

In some embodiments of the first, seventh or ninth aspect, a kit maycomprise a remineralisation agent, or the method may compriseremineralising the tooth or sample thereof. A remineralisation agent maycomprise fluoride, soluble calcium phosphate or amorphous calciumphosphate, which may be stabilised with bioactive molecules.

EXAMPLES Example 1 The Protein Composition of MIH Enamel Depends onSurface Integrity Materials and Methods Specimens

Human and Sprague-Dawley rat specimens were obtained with appropriateethical approvals, and stored at −80° C. MIH was diagnosed according tostandard criteria (Weerheijm, 2003). After extraction, MIH teeth werewater-rinsed to remove visible blood, then blotted dry and stored frozenimmediately. Whole saliva, stimulated by chewing on wax, was clarifiedby centrifugation (20,000 g, 5 min) before storage. Serum anderythrocytes were prepared conventionally from blood of 6-day-old rats.Secretory enamel matrix was isolated from developing rat teeth as before(Hubbard, 1996) except using 5-day-old first molars.

Profiling of Enamel Proteins

Overt MIH lesions were collected from freshly thawed specimens byscraping with a scalpel, taking care to avoid carious enamel anddentine. Normal enamel was sampled using a slowly rotating dental bur(No. 6). Immediately afterwards, enamel samples (2-5 μl packed vol) weresuspended in 10% trifluoroacetic acid (10 volumes, 10 min at roomtemperature with vortexing and bath sonication), then centrifuged(20,000 g, 4° C., 5 min) to sediment acid-insoluble protein. Pelletswere solubilized in gel-loading buffer containing 2% SDS and 100 mmol/Ldithiothreitol (Hubbard, 1996), with additional protease inhibitors (1mmol/L phenylmethylsulfonyl fluoride, 1 mmolIL benzamidine, 5 μg/mLpepstatin, 5 μg/mL leupeptin) where indicated. SDS extracts werequantified by dot blotting with Amido Black and subjected to miniSDS-PAGE with Coomassie Blue staining or immunoblotting (Hubbard, 1995).Amelogenin antiserum was raised conventionally in rabbits, usingrecombinant mouse amelogenin (SEQ ID NO: 19) as immunogen.

Proteomics Analysis

Gel bands were subjected to trypsinolysis and tandem mass spectrometryas before (Mangum et al., 2006) except using an ion-trap instrument withchip-based nanospray (Chip-LC/MSD XCT, from Agilent Technologies, SantaClara, Calif., USA). Proteins were identified using the MASCOT searchengine and SwissProt human database with strict acceptance criteria(minimally two sequence tags (Mangum et al., 2006)).

Mineral-Binding Assays

Mock oral fluid was prepared by empirically spiking saliva with serumand erythrocyte lysate so that major proteins from all three componentswere similarly abundant (FIG. 4B). To assay protein binding, oral fluidwas incubated with 0.1 volumes hydroxyapatite (from Sigma, St Louis,Mo., USA) or MIH enamel for 60 min at 20° C. then centrifuged (2,000 g,2 min). After washing in 3 volumes 20 mM Tris-HCl (pH 8.0), the pelletwas extracted with trifluoroacetic acid and SDS as described above forenamel.

Results

MIH Enamel is Enriched with Non-Amelogenin Proteins

Profiling of enamel proteins has provided useful insights to thepathogenesis of fluorosis and amelogenesis imperfecta, particularly bylinking amelogenin levels with clinical properties. Accordingly, unfixedMIH enamel specimens were investigated using an SDS-PAGE approach.Unlike normal enamel, MIH enamel gave visible precipitates whendissolved in acid, suggesting a relatively high protein content. Asshown in FIG. 1, quantification of acid-insoluble protein from fivesevere lesions yielded values 3- to 15-fold higher than normal (0.3-1.5%protein w/w). Similarly, SDS-PAGE with Coomassie staining revealednumerous protein bands in MIH enamel contrasting with barely detectablebanding in normal enamel (FIG. 2A). Since amelogenins were undetected(FIG. 2A, 20-25 kDa region), immunoblotting was used for highersensitivity. Anti-amelogenin also failed to detect intact amelogenins inMIH enamel, but degradative fragments were observed in some specimens(FIG. 2B, specimen 11). Amelogenins were undetectable in normal enamelunder these conditions (not shown). Quantitative comparison withsecretion-phase enamel matrix showed that MIH enamel contained only0.12%±0.06% (±SE, n=6) the amount of total detectable amelogenins (FIG.2B, 8- to 25-kDa region). It was concluded that MIH enamel isprotein-enriched, and for pathogenic reasons other than amelogeninretention.

Body Fluid Proteins Predominate in Mih Enamel

To identify the major protein constituents of MIH enamel, SDS-PAGE bandswere subjected to proteomic analysis. As shown in FIG. 3 and Table 1, avariety of proteins were identified (16 distinct gene products), 13 ofwhich are found in saliva and crevicular fluid. The three others(haemoglobin, albumin, complement C3) are major components of blood.Consequently all major proteins identified in MIH enamel are normallyassociated with body fluids found intraorally.

TABLE 1 Proteins identified in MIH enamel with intact surface (specimens1 to 6) and with post-eruptive breakdown (specimens 7 to 11) SpecimensMASC Name Body fluid Mass (kDa) identified Peptides Coverage OT (UniProtacc.) localization Observed Theoretical in (n) (%) Score Serum albumin(P02768) Serum, saliva, 70 69 1 16 21 426 GCF 3 2 4 89 4 15 26 471 5 1021 197 6 9 15 389 10 7 14 212 3 3 8 87 60 5 7 18 323 6 12 15 389 10 1424 461 10 2 5 47 40 10 3 2 50 32 7 5 8 136 10 Complement C3 beta chainSerum 70 71 7 2 1 165 (P01024) Alpha-1-antitrypsin Serum, saliva, 40 447 14 33 308 (P01009) GCF 25-30 7 3 16 122 Protein S100-A9 Saliva, GCF25-30 13 7 3 24 54 (P06702) 13 7 5 37 212 13 11 6 56 158Lactotransferrin (P02788) Saliva, GCF 70 78 11 2 4 155 Leukocyteelastase inhibitor Blood, saliva 40 43 7 4 9 172 (P30740)Antithrombin-III (P01008) Serum, saliva 40 53 7 2 4 121 Hemoglobinsubunit alpha Blood, saliva 13 15 7 6 38 117 (P69905) 13 11 5 23 115Hemoglobin subunit beta Blood, saliva, 13 16 7 12 63 374 (P68871 ) GCF13 10 2 12 51 13 11 11 63 207 Hemoglobin subunit delta Blood, saliva 1316 7 8 40 190 (P02042) 13 11 6 50 145 Prolactin-inducible protein Saliva13 17 11 2 15 85 (P12273) Alpha amylase 1 (P04745) Saliva 60 57 8 2 7131 Ig kappa chain V-III region Blood, saliva 25-30 12 7 2 16 130 SIE(P01620) 25-30 8 2 16 67 25-30 11 2 16 91 Ig alpha-2 chain C regionBlood, saliva 60 37 10 3 7 109 (P01877) Uncharacterized protein Saliva32 38 10 4 14 68 c6orf58 (Q6P5S2) Serpin B3 (P29508) Blood, saliva 27 4510 2 4 48Intact and Broken MIH Lesions have Distinct Protein Profiles

Given the clinical diversity of MIH lesions (colour, consistency, size,surface integrity), it was investigated whether the differentpresentations have distinct protein compositions. Appraisal of theprotein profiles (FIG. 2A) led to the hypothesis that integrity of theenamel surface had a major influence. Notably, when lesions were groupedas “intact” and “broken”, the protein-banding patterns appearedqualitatively similar within each group, but two striking differenceswere apparent between the groups (FIG. 2A, 12-kDa & 66-kDa regions). The12-kDa band, which was obvious in broken but not intact lesions,routinely contained haemoglobin as a major component (FIG. 3).Conversely, in intact lesions the 66-kDa band routinely containedalbumin only, unlike broken lesions where albumin was found infrequentlyat lower levels.

The stability of the protein profiles was also queried, noting evidenceof protein degradation (FIG. 3: albumin, complement C3) and the key roleof proteolysis in enamel maturation. Indeed, when SDS-solublized samplesfrom FIG. 2A were reanalysed after frozen storage, the albumin bands hadcompletely disappeared from intact specimens (FIG. 2C). Broken specimenswere largely unaffected however (not shown). Protease inhibitors hadlittle effect on the profiles of fresh MIH samples when added during theinitial SDS-solubilisation step (not shown). These results highlightedthe risk of artefactual proteolysis and hence only first-run samples arereported (FIGS. 1 to 3). It was concluded that intact and broken lesionsconsistently have distinct protein profiles, supporting the hypothesisthat surface integrity influences the protein composition of MIH enamel.

Protein Composition of MIH Enamel Varies with Surface Integrity

It is known that MIH lesions exhibit sub-surface porosity and thatalbumin and haemoglobin bind avidly to hydroxyapatite. Accordingly, itwas posited that oral-fluid proteins permeate MIH enamel and selectivelybind to hydroxyapatite crystals, subject to absence of an intact surfacelayer. When broken lesions were compared with saliva, serum anderythrocytes, collective similarities in the protein-banding patternswere found (FIG. 4A). In contrast, intact lesions bore an intriguingresemblance to serum alone. These results accorded with oral-fluidproteins being excluded from intact but not broken lesions. Next, abroken lesion was modelled by exposing hydroxyapatite powder to mockoral fluid (combination of saliva, serum and erythrocyte extract).Profiling of the hydroxyapatite-bound fraction (FIG. 4B) revealedremarkable similarity to broken lesions (FIGS. 2A, 4A). Whenhydroxyapatite was substituted with powdered enamel made from an intactlesion (i.e. to model breakage of the surface layer), the profile wasagain similar to broken lesions (FIG. 4C). These results indicated thatthe protein composition of MIH enamel is strongly influenced byintegrity of the enamel surface.

When mock oral fluid comprising albumin and hemoglobin was applied tohydroxyapatite, albumin and hemoglobin bound to the hydroxyapatite (FIG.5). Washing sequentially in each of 5 mM MgCl₂, 1 M MgCl₂, and 0.4 MNaH₂PO₄ each for 5 min removed >90% of protein from hydroxyapatite (FIG.5).

Discussion

Given growing concerns about MIH worldwide, a pressing need exists toelucidate the protein composition of hypomineralized enamel. It isdisclosed herein that MIH enamel has substantially higher proteincontent than normal, but a near-normal level of residual amelogenins.This characteristic distinguishes MIH from hypomaturation defects thatcontain high residual amelogenins (amelogenesis imperfecta, fluorosis)and in turn typifies MIH as a hypocalcification defect. Secondly, MIHenamel was found to have accumulated various proteins from oral fluidand blood, with differential incorporation depending on integrity of theenamel surface. Pathogenically, these results point to a pre-eruptivedisturbance of mineralisation involving albumin and, in cases withpost-eruptive breakdown, subsequent protein adsorption to the exposedhydroxyapatite matrix. These insights to the pathogenesis and propertiesof MIH enamel hold significance for the prevention, diagnosis andtreatment of MIH.

The present results help to explain the clinical and biophysicalproperties of MIH enamel. The observed 3-fold to 15-fold elevation inprotein content is similar to reports for amelogenesis imperfecta andfluorosis (2.5-fold to 30-fold) and appears sufficient to account forthe characteristic mechanical weakness of MIH enamel. The low residualcontent of amelogenins likens MIH enamel to hypocalcified types ofamelogenesis imperfecta. Enamel from the latter disorders is describedclinically as markedly softer than normal and friable or cheesy, whichcoincides with descriptions of MIH enamel. At the protein level, MIHenamel appears distinguishable from hypocalcified types of amelogenesisimperfecta and fluorosis, particularly based on its uniquely highcontent of albumin. However, all conditions are characterised by poroushydroxyapatite.

These results also elucidate the pathogenesis of MIH, pointing to pre-and post-eruptive steps that are mechanistically distinct.Pre-eruptively, the normal thickness and low amelogenin content of MIHenamel (<0.2% of secretion-phase level) indicates that amelogenins aresecreted and then removed effectively. It follows that MIH is not amaturation defect primarily. By analogy to hypocalcified amelogenesisimperfecta, attention therefore turns to defective initiation ofmineralisation. Protein profiling indicated that albumin accumulates inMIH enamel despite near-complete removal of amelogenins.

In other words, hypocalcification is a subtype of hypomineralisation,the other subtype being hypomaturation. As shown herein, MIH and sometypes of amelogenesis imperfecta, and probably some types of fluorosistoo, are distinguished as hypocalcification defects in that they havelow amounts of amelogenin. That is, the normal process of amelogeninremoval (enamel maturation) has occurred, but calcification has notoccurred. In hypomaturation defects, however, (immature types ofamelogenesis imperfecta and fluorosis), amelogenin removal (enamelmaturation) has not occurred to a major degree and it is the continuedpresence of amelogenin that impedes calcification.

Amelogenin levels are relatively low in hypocalcification types ofDDD/hypomineralisation, but closer to normal levels in hypomaturationtypes of DDD/hypomineralisation (such as some types of amelogenesisimperfecta and dental fluorosis). Therefore, variations in levels bothof amelogenin and the remaining proteins disclosed herein bound toporous dental hydroxyapatite could be informative (e.g. diagnostic)individually or in combination, for example as a ratio.

For the first time, these results demonstrate extravasated albumin beingaccumulated in malforming human enamel. Notably, intact lesions werefound to contain albumin but not numerous oral-fluid proteins withdemonstrated hydroxyapatite-binding potential. That albumin but nothaemoglobin was prominent may be attributed either to a minor vascularleak of serum rather than whole blood, or to high proteolytic stabilityof albumin relative to haemoglobin and other blood proteins duringenamel maturation. Indeed, albumin is resistant to kallikrein-relatedpeptidase 4, the major protease implicated in amelogenin removal.

These results also imply that another pathogenetic step followspost-eruptive breakdown of the enamel surface. This second step involvesrelatively promiscuous binding of oral-fluid proteins to the exposedhydroxyapatite matrix.

The proteins identified herein have potential utility as biomarkers forcharacterizing MIH lesions clinically.

Example 2 Production and Testing of a Probe for Porous HydroxyapatiteMaterials and Methods

SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexanecarboxylic acidN-hydroxysuccinimide ester; CAS#: 64987-85-5) is a non-cleavableheterobifunctional cross-linker with amine and sulfhydryl reactivityseparated by a spacer arm of 8.3 Å. Amido black (CAS#: 1064-48-8) is acommon blue/black stain used here as a coloured reporter which containsa primary amine group. Hemoglobin from cow (CAS#: 9008-02-0) is aheterotetramer consisting of 2 pairs of polypeptide chains (α and β; SEQID NOs: 20 and 21, respectively). The β-chain has a singlesolvent-exposed sulfhydryl-containing cysteine residue, while theα-chain has no cysteines.

SMCC (75 mM in dimethyl sulfoxide) was added to 9-volumes of amido black(37.5 mM in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10mM sodium phosphate dibasic, pH 7.4)) and incubated 30 minutes at 21° C.The 5-fold molar excess of Amido black ensured maximal labelling of SMCC(creating a maleimide-activated coloured reporter, FIG. 27). Afterconjugation, the solution was desiccated by vacuum centrifugation andstored at −80° C.

Hemoglobin (20 mg/ml; 0.65 μmole cysteine-thiol/ml) was prepared bydissolving in PBS that contained 10 mM TCEP(tris(2-carboxyethyl)phosphine, a non-thiol reducing agent used tomaintain cystine-sulfhydryl state) and 5 mM EDTA(ethylenediaminetetraacetic acid, a metal chelator used to reducepotential for oxidant/radical catalysis and subsequent thiol oxidation).After a 30 minute incubation at 21° C., reduced hemoglobin was dialysedagainst 1,000-volumes of PBS for 2 hours to deplete TCEP and EDTA (thisstep may be optional). The hemoglobin was taken to the next stepimmediately to minimise cysteine-thiol oxidation.

The desiccated maleimide-activated Amido black was dissolved inHemoglobin at a Amido black:thiol molar ratio of 10:1 to ensure maximallabelling of hemoglobin. After incubating for 2 hours at 21° C., Amidoblack-conjugated hemoglobin was dialysed extensively against PBS (untildialysate remained uncoloured, for 1 ml this took 24 to 48 hours) toremove non-covalently bound amido black. After dialysis, the probe wasready for use.

Results

Within 5 min of applying the probe, hydroxyapatite changed in colourfrom white to dark blue (FIG. 29). The probe withstood washing in water,whereas Amido black alone (i.e. not linked to Hb) was removed by washingin water. The probe was removed from hydroxyapatite with a three-stepwashing procedure that comprised washing sequentially in each of 5 mMMgCl₂, 1 M MgC12, and 0.4 M NaH₂PO₄ for 5 min (FIG. 29).

Discussion

A key design requirement was the preservation of hemoglobin'shydroxyapatite-binding function after conjugation to the colouredreporter. Hemoglobin's cysteine-thiols were targeted because two of thefour protein subunits carry a single cysteine (not at bindinginterfaces); the other two subunits lack cysteine. The resultingtetramer probe therefore contains two unmodified protein subunits,thereby maintaining at least half the native hydroxyapatite-bindingsites per functional unit. A 2-step method was exemplified: the firstproduced a coloured reporter-SMCC conjugate (FIG. 27); the second usedthe coloured reporter-SMCC conjugate to label hemoglobin (FIG. 28). Hereproof-of-principle has been established for design, production andtesting of a novel probe that detects porous hydroxyapatite.

Example 3 The Probe Binds to Porous Dental Enamel Specifically (FIG. 30)Methods

To test whether the probe of Example 2 binds to porous enamelspecifically, a complex carious lesion was coated with the probe thenwashed thoroughly.

A human first molar that had a large region of caries (porous enamel,white opaque region) was photographed before and after application ofthe probe (FIG. 30). The probe was applied to the whole crown regionusing a brush for a period of one minute. After application of theprobe, the tooth was rinsed under running water for 10 seconds,photographed, then the tooth was washed again for a further two minutesand photographed.

Results

Normal enamel was not labelled.

Regions of overt caries were labelled strongly and specifically, butlabelling was patchy in some places. The unlabelled carious regionsexhibited a shiny surface that was resistant to scratching, whereaslabelled regions had a dull surface that could be scratched readily.This indicates that the areas of patchy unlabelled caries may be due toremineralisation of the surface layer. Thus, the probe is capable ofdiscriminating between active and inactive caries.

Regions of enamel broken during extraction of the tooth (forcepsimprints) were also labelled indicating that the probe can detectregions of enamel that have a breached surface.

The probe provided a stable level of labelling, independent ofwater-rinsing time.

Example 4 The Probe Can Specifically Detect Early Demineralisation ofSurface Enamel (Model of Incipient Caries) (FIG. 31) Methods

To test whether the probe of Example 2 can specifically detect earlycaries, artificial carious lesions were produced on normal surfaceenamel using spots of strong acid (before application of probe).

A human first molar was shown by photography before and afterapplication of the probe to be caries-free prior to acid-treatment (FIG.31). Three regions of enamel were then exposed to acid (0.5 μl 85%H₃PO₄) for 1, 3 or 10 minutes to introduce artificial carious lesionsbefore washing in 100 ml TBS (25 mM Tris pH 7.2, 160 mM NaCl) for twominutes, then under running water for another two minutes. The tooth wasair-dried and the probe was applied to the whole area for three minutesusing a brush. After application, unbound probe was removed by firstwiping with absorbent paper, then by rinsing under running water for 10seconds. To remove bound probe, 10% bleach (0.4% NaClO) was applied witha brush for 10 seconds.

Results

The probe did not bind to any regions of the caries-free enamel.

Acid etch treatment yielded three regions of slightly opaque/dullenamel, which followed a dose-dependent severity profile (10>3>1 min).The three etched regions were all detected by the probe, in aseverity-dependent manner; un-etched enamel was not labelled.

Probe binding resisted washing in water, although signal intensitydiminished slightly. The probe could be quantitatively removed byapplication of 10% bleach for 10 seconds.

Example 5 Probe's Mechanism of Action is Hydroxyapatite Affinity (FIG.32)

Enamel from Example 4 (FIG. 31) was re-treated with probe of Example 2to verify a hydroxyapatite-binding mechanism.

Methods (A)

To rule out a protein-staining mechanism, the probe was applied toetched enamel that had been bleach-treated (i.e. protein stripped).

Results (A)

Bleached enamel was labelled by the probe similarly to unbleached (FIG.32, compare Panels A2 and A4). This finding rules out a protein-stainingmechanism for the probe's labelling of etched enamel.

Methods (B)

It was proposed that, if the probe's mechanism of action ishydroxyapatite-binding, then BSA pre-treatment should block probebinding (competitive inhibition).

Enamel from Panel A was exposed to a known hydroxyapatite-bindingprotein (10% bovine serum albumin, BSA) by applying with a brush for oneminute, followed by water rinsing for one minute. After BSA treatment,the probe was applied as before. BSA was stripped by bleach treatment,and the probe re-applied.

Results (B)

Application of BSA did not alter appearance of the enamel (FIG. 32,Panel B2). BSA blocked binding of the probe (FIG. 32, Panel B3). Probebinding was restored after stripping BSA (FIG. 32, Panel B6). Together,these results demonstrate that the probe's mechanism of action ishydroxyapatite-binding, not protein-binding. Given the possibility ofcompetitive inhibition by other hydroxyapatite-binding proteins,pre-treatment to strip proteins could improve probe sensitivity and sominimise false-negative results.

Example 6 The Probe Specifically Labels Hypomineralised Enamel andAbnormal Dentine (FIG. 33) Methods

To test whether the probe of Example 2 could be used to delineateabnormal dental tissues, a portion of tooth that contained normal andabnormal enamel & dentine was treated with the probe.

A fractured tooth that displayed a region of sub-surfacehypomineralisation was chosen to mimic a clinically difficult case wherelesion boundaries are obscure and complex. A brief pre-exposure to theprobe led to demarcation of the enamel-dentine boundary. The specimenwas then photographed before (left) and after (right) the probe wasapplied with a brush for 30 seconds (FIG. 33). After application,unbound probe was removed by rinsing in water for 30 seconds.

Results

Before application of the probe, several structures could be identified:(1) normal enamel which overlaid (2) hypomineralised enamel (pink incolour with a red border in some regions), (3) apparently normal dentine(hard) and (4) abnormal dentine (soft/leathery). After application ofthe probe, all four types of tissue could be readily discerned.

Normal enamel and dentine were unlabelled. Hypomineralised enamel wasuniformly and specifically labelled an intense violet colour, whichappeared to trace a very complex border throughout the subsurfaceregion. Abnormal dentine (potentially due to caries and/or developmentaldefects) was specifically and uniformly labelled a deep green colour,which appeared to trace complex borders against normal dentine.Together, these data confirm that the probe can specifically labelhypomineralised enamel and abnormal dentine.

Example 7 The Probe Can be Used to Guide Removal of HypomineralisedEnamel (FIG. 34) Methods

Hypomineralised enamel from Example 6 (FIG. 33) was removed using ascalpel blade and repeatedly re-probed with the probe of Example 2 tomonitor progress. Physical characteristics of the enamel were noted ateach step (FIG. 34, see description beneath panels).

Results

The upper panels of FIG. 34 show the specimen after removal ofhypomineralised enamel, whereas the lower panels show the same specimenafter application of the probe. Panels 1 to 3 show gradual removal ofsmall regions of hypomineralised enamel. Panels 4 to 6 show attemptedremoval of the whole region, and regions of incomplete removal (compareupper and lower panels). Note that as hypomineralised enamel wasremoved, the physical characteristics changed markedly in parallel withdegree of labelling, to the end-point where remaining enamel wasphysically uniform and unstained by the probe (Panel 6).

Abnormal dentine was not addressed in this example.

Example 8 The Probe can be Used to Guide Removal of Abnormal Dentine(FIG. 35) Methods

Abnormal dentine from Example 6 (FIG. 33) was removed using a scalpelblade and iteratively re-probed with the probe of Example 2 to monitorprogress of removal. Physical characteristics of the dentine were notedat each step (FIG. 35, see description beneath panels).

Results

The upper panels of FIG. 35 show the specimen after removal of abnormaldentine, whereas the lower panels show the same specimen afterapplication of the probe. Panel 1 shows intense staining of abnormaldentine, which is reduced sharply with removal and reprobing (e.g.compare lower panels 1 and 2). Reduced levels of labelling by the probecorrelate with improved physical character of the dentine (e.g. in Panel4, the dentine hardness was uniformly normal by physical assessment andlargely unstained after application of the probe). Note that even aftercomplete removal of abnormal dentine, a low level of background stainingis apparent (presumably due to dentine's higher porosity relative toenamel).

Example 9 Detection of Abnormal Dentine by the Probe Can be Improved bya Bleach Wash (FIG. 36) Methods

To test whether the probe's specificity for dentine, as shown in Example8, could be improved, a bleach wash was used to reduce staining ofnormal dentine.

A human molar with exposed normal and abnormal dentine was exposed tothe probe of Example 2 (brush application for one minute followed bywater rinse for one minute) and subsequently exposed to a bleach wash(applied with brush for 10 seconds, then water rinsed for one minute).Following probe/bleach application, labelled regions were removed with ascalpel blade then re-probed/bleached to monitor progress (FIG. 36).

Results

Abnormal dentine was preferentially detected by the probe, howeverbackground staining of normal dentine decreased confidence in borderdemarcation. Application of 10% bleach (0.4% NaOCl) for 10 secondsimproved resolution by reducing labelling in normal dentine, but not inabnormal. Application of neat bleach (4% NaOCl) for 10 secondscompletely removed labelling from normal dentine, without affectinglabelling of abnormal dentine, resulting in much clearer delineation ofabnormal dentine.

After neat bleach, abnormal dentine was removed (Panel 4) thenre-probed/bleached (Panel 5), showing that most, but not all, abnormaldentine was removed. Another removal/re-probe/bleach step showed thatabnormal dentine was completely removed. The remaining dentine wasphysically indistinguishable from normal dentine.

Together, these results suggest that a protein-stripping step afterapplication of the probe can help reduce background labelling of normaldentine, reducing potential false-positive readouts.

Example 10 The Probe can be Opaque to X-Rays (FIG. 37) Methods

The probe was made radio-opaque by substituting the blue chromophore(amido black) of Example 2 for 5-amino-2,4,6-triiodoisophthalic acid(³I), a precursor compound used in medical radiography (e.g. forcerebral angiography). This compound was chosen due to the availabilityof a single primary amine that could be used for coupling with the samecross-linker used in the blue probe.

To couple ³I to hemoglobin, the following procedure was used:

1. 1.25 mg of SMCC (cross-linker) was dissolved in 50 μl DMSO (75 mMSMCC).

2. ³I was prepared as follows: 30 mg was dissolve in 1 ml 0.1M NaOH (50mM ³I), 250 μl 0.1 M HEPES pH 7.0 was added, then pH was adjusted to 7with 1 μl additions of 5 M NaOH; such that the final solution was 40 mM³I, 20 mM HEPES pH 7.

3. 400 μl of ³I solution was added to 50 μl 75 mM SMCC in DMSO andincubated at room temperature for 30 minutes to generate ³I-activatedSMCC.

4. The ³I-SMCC was then lyophilised by vacuum centrifugation.

5. The resultant pellet was taken up in 20 μl of DMSO and 100 μl of 20mg/ml hemoglobin was added, then the solution was incubated at roomtemperature for 60 min to couple ³I to cysteine thiols in hemoglobin.

6. The resultant ³I-Hb was then centrifuged (20,000×g for 5 minutes)before dialysis (10-kDa MWCO) overnight against 25 mM Tris pH 7.2, 160mM NaCl.

7. The resulting dialysate was collected and stored at −20° C.

To assess the degree of radio-opacity conferred on the probe, it wassubjected to X-ray radiography (65 kV, 8 mA, 0.5 second exposure)alongside radio-opaque standards (1 and 10 mM ³I).

Results

The X-ray probe was radio-opaque to a degree between 1 and 10 mM ³I(FIG. 37). Density analysis suggested radio-opacity was equivalent to a1.5-2.5 mM solution of ³I. These results confirm that the probe can bemade opaque to X-rays.

Example 11 Analysis of Washing Solutions Using Pure Hydroxyapatite (FIG.38) Methods

To examine the relative effectiveness of each wash solution, they wereindividually tested using an in vitro model system (FIG. 38).

Pure hydroxyapatite (5 mg) was loaded with proteins from rat blood (100μl of 10 mM Tris pH 7.2 which contained 10 μl Hb extract and 2 μl neatserum) for 10 minutes at room temperature with constant shaking.Protein-loaded hydroxyapatite was sedimented by centrifugation at2,000×g for 30 seconds, the supernatant was discarded then the pelletwas washed with 300 μl 10 mM Tris pH 7.2 for 30 seconds to removeunbound interstitial components.

Protein-hydroxyapatite was then exposed to 100 μl of various washcomponents (water, 5 mM MgCl₂, 1 M MgCl₂ or 0.4 M NaH₂PO₄) for 2 minutesat room temperature with mixing before centrifugation. Washes werecollected and Protein-hydroxyapatite was washed another two times withthe same washing solution. After three wash steps, theProtein-hydroxyapatite was dissolved in 100 μl 10% trifluoroacetic acid(TFA), and precipitated proteins collected by centrifugation (2,000×gfor 2 minutes), and pellets were dissolved in 100 μl of 2×SoB (0.125MTris-HCl pH 6.8, 4% SDS, 20% Glycerol). Protein content in all fractionswas assessed by densitometry of dot-blots stained with Amido Black.

Results

The relative capabilities of the washing solutions to remove proteinfrom hydroxyapatite were:

0.4 M PO₄>1M Mg²⁺>5 mM Me⁺(no more effective than water).

Although PO₄ appeared to provide the best protein-removal, is was notedthat the hydroxyapatite remained a pink colour even after 3 washes,whereas the 1M Mg²⁺-treated hydroxyapatite became white after a singlewash. This being the case, it appears 1M MgCl₂ and 0.4M NaH₂PO₄ are mosteffective at removing protein, and they have complementary activities(likely removing different classes of proteins).

Example 12 Analysis of Mg²⁺and PO₄ Separately or Sequentially Using PureHydroxyapatite (FIG. 39) Methods

Pure hydroxyapatite was loaded with proteins, then subjected to 100 μlof various wash components (water, 1 M MgCl₂ or 0.4 M NaH₂PO₄) as forExample 11, except with two washes (instead of three) and 5 mM MgCl₂ wasomitted. One tube received 1M Mg²⁺ followed by 0.4 M PO₄. After thewashes, hydroxyapatite pellets were photographed to record the colour(see inset), then protein content was assessed as for Example 11.

Results

All three washing solutions performed similarly, removing the majorityof proteins after two washes, unlike water (FIG. 39).

Sequential washing with Mg²⁺ then PO₄ produced the best result asassessed by protein removal and colour removal (inset: arrows indicatehydroxyapatite pellets after washing). It may be concluded thatsequential washing with Mg²⁺and PO₄ provide optimal protein removal, inthis hydroxyapatite model.

Example 13 Analysis of Combined Mg²⁺Plus PO₄ Wash Using PureHydroxyapatite (FIG. 40) Methods

Pure hydroxyapatite was loaded with protein, then subjected to 100 μl ofcombined wash (1 M MgCl₂, 0.4 M NaH₂PO₄) three times as for Example 11.Protein content was assessed as for Example 11. Note that results fromExample 13 (FIG. 40) are charted alongside data from Example 12 forcomparison.

Results

The combination wash performed similarly to PO₄ alone, however thehydroxyapatite turned white after the first wash (similar to 1MMg²⁺alone), indicating the activity of each wash component was retained.It may be concluded that a combined wash may be more effective in termsof the time required to achieve protein removal.

Example 14 Washing Solutions Work on Hypomineralised Enamel, Althoughwith Reduced Efficacy Compared With the Hydroxyapatite Model (FIG. 41)Methods

Hypomineralised enamel from intact and broken lesions was collectedseparately such that 3 tubes of 5 mg powder were available for each typeof lesion. Enamel was exposed to 100 μl of 5 mM Mg²⁺, 1 M Mg²⁺, then 0.4M PO₄, each for 5 minutes. Samples were then treated as for the purehydroxyapatite of Examples 11 to 13.

Results

Treatment of hypomineralised enamel with washing solutions removed asubstantial amount of protein (˜¼-⅓), whereas water was barely effective(FIG. 41). The amount of protein removed was less that that seen for thehydroxyapatite model, possibly due to slower off-rates.

Example 15 Washing Solutions Can Quantitatively Remove Proteins fromHypomineralised Enamel (FIG. 42) Methods

Hypomineralised enamel from an intact lesion was collected such that 3tubes of 5 mg was available. Enamel was exposed to 1 ml of 1 M Mg²⁺ for7 hours, then 1 ml 0.4 M PO₄ for a further 16 hours. Samples were thentreated as for the pure hydroxyapatite of Examples 11 to 13.

Results

Proteins were quantitatively removed from hypomineralised enamel aftertwo extended washes with washing solutions (FIG. 42B), whereas watertreatment over the same timeframe had little effect. The PO₄ wash hadgreatest effect, likely due to the protein profile of this particularlesion (predominantly albumin, FIG. 42A). While the timeframe may belonger than desirable, the washing solutions are capable of removing allprotein from clinical specimens.

REFERENCES

-   Hubbard M J (1995). Calbindin 28 kDa and calmodulin are    hyperabundant in rat dental enamel cells. Identification of the    protein phosphatase calcineurin as a principal calmodulin target and    of a secretion-related role for calbindin28 kDa. Eur J Biochem    230:68-79.-   Hubbard M J (1996). Abundant calcium homeostasis machinery in rat    dental enamel cells. Up-regulation of calcium store proteins during    enamel mineralisation implicates the endoplasmic reticulum in    calcium transcytosis. Eur J Biochem 239:611-623.-   Mangum J E, Veith P D, Reynolds E C, Hubbard M J (2006). Towards    second-generation proteome analysis of murine enamel-forming cells.    Eur J Oral Sci 114 Suppl 1:259-265.-   Weerheijm K L (2003). Molar incisor hypomineralisation (MIH). Eur J    Paediatr Dent 4:114-120.

1-51. (canceled)
 52. A kit, when used for detecting porous dentalhydroxyapatite, comprising: a protein capable of binding porous dentalhydroxyapatite; or a detector that detects said protein bound to porousdental hydroxyapatite, optionally wherein the protein is selected fromthe group: Serum albumin; Complement C3 beta chain; Alpha-1-antitrypsin;Protein S100-A9; Lactotransferrin; Leukocyte elastase inhibitor;Antithrombin-III; Hemoglobin subunit alpha; Hemoglobin subunit beta;Hemoglobin subunit delta; Prolactin-inducible protein; Alpha amylase 1;Iv kappa chain V-III region SIE; Ig alpha-2 chain C region;Uncharacterized protein c6orf58; Serpin B3; and Amelogenin. 53.(canceled)
 54. The kit of claim 52, wherein the protein specificallybinds porous dental hydroxyapatite.
 55. (canceled)
 56. The kit of claim52, further comprising a reporter that specifically binds to or islinked to the protein or detector.
 57. The kit of claim 56, wherein thereporter is colored or radio-opaque.
 58. The kit of claim 57, whereinthe colored reporter is amido black.
 59. (canceled)
 60. The kit of claim56, further comprising a linker for linking the reporter to the proteinor detector.
 61. The kit of claim 60, wherein the linker is aheterobifunctional cross-linker.
 62. (canceled)
 63. The kit of claim 52,wherein the protein is Hemoglobin or a subunit thereof.
 64. (canceled)65. A probe, when used for detecting porous dental hydroxyapatite,comprising: a protein capable of binding porous dental hydroxyapatite;and a reporter, optionally wherein the protein is selected from thegroup: Serum albumin; Complement C3 beta chain; Alpha-1-antitrypsin;Protein S100-A9; Lactotransferrin; Leukocyte elastase inhibitor;Antithrombin-III; Hemoglobin subunit alpha; Hemoglobin subunit beta;Hemoglobin subunit delta; Prolactin-inducible protein; Alpha amylase 1;Ig kappa chain V-III region SIE; Ig alpha-2 chain C region;Uncharacterized protein c6orf58; Serpin B3; and Amelogenin. 66.(canceled)
 67. The probe of claim 65, wherein the protein is Hemoglobinor a subunit thereof.
 68. The probe of claim 65, wherein the reporter iscolored or radio-opaque.
 69. The probe of claim 68, wherein the coloredreporter is amido black.
 70. (canceled)
 71. The probe of claim 65,wherein the probe further comprises a linker linking the protein to thereporter.
 72. (canceled)
 73. The probe of claim 71, wherein the linkeris a heterobifunctional cross-linker. 74-75. (canceled)
 76. A method fordetecting a condition involving porous dental hydroxyapatite, comprisingdetecting in or on a tooth or a sample of the tooth of a subject aprotein bound to porous dental hydroxyapatite, optionally wherein theprotein is selected from the group: Serum albumin; Complement C3 betachain; Alpha-1-antitrypsin; Protein S100-A9; Lactotransferrin; Leukocyteelastase inhibitor; Antithrombin-III; Hemoglobin subunit alpha;Hemoglobin subunit beta; Hemoglobin subunit delta; Prolactin-inducibleprotein; Alpha amylase 1; Ig kappa chain V-III region SIE; Ig alpha-2chain C region; Uncharacterized protein c6orf58; Serpin B3; andAmelogenin. 77-78. (canceled)
 79. The method of claim 76, wherein thedetecting comprises immunodetection, chromatography, electrophoresis, ormass spectrometry.
 80. The method of claim 76, wherein the subject is ahuman.
 81. The method of claim 80, wherein the human is under about 12years of age, about 2 to about 12 years of age, about 4 to about 10years of age, or about 6 to about 10 years of age.
 82. The method ofclaim 76, further comprising permeabilizing the tooth or a sample of thetooth before detecting the protein.
 83. The method of claim 76, whereinthe condition is dental caries, Molar/Incisor Hypomineralization (MIH),amelogenesis imperfecta, dental fluorosis, or other developmental dentaldefect (DDD). 84-102. (canceled)